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| Copy Number Analysis of Lipoxygenase RNAi Gene (Loxi) in Transgenic Lines ofWheat (Triticum aestivum) |
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Abstract The copy number of exogenous gene in transgenic plants is an important factor influencing the expression and genetic stability of transgene. To detect the copy number of exogenous target gene RNAi sequence of lipoxygenase gene (Loxi) in 6 transgenic lines of wheat (Triticum aestivum) from 2 populations (3 transgenic lines in from each population), and analyze the activity of lipoxygenase in seeds and obtain transgenic wheat lines with low lipoxygenase activity and stable inheritance of Loxi, TaqMan Real-time quantitative PCR was performed to estimate the copy number of Loxi in wheat transgenic TF5 lines, using an endogenous single copy gene Pinb (puroindoline b) as a reference gene. The lipoxygenase activity was determined using the linoleic acid as the substrate, and statistical analysis was performed simultaneously. The results showed that correlation coefficients of standard curve for the reference gene and the exogenous gene both were close to 1, and the amplification efficiency of Pinb and Loxi genes were appropriately, which was 107.7% and 104.5%, respectively. The copy number of Loxi in 6 transgenic wheat lines were different, the minimum and maximum copy number was 1 and 8, respectively. The lipoxygenase activity in the seeds of 2 parent cultivars Shanyou 225 and Xinong 889 were (860±28.20) and (1 132±59.80) U, respectively. Compared to that of their parent materials, the lipoxygenase activities in the seeds of transgenic lines were decreased by various degree (approximately from 21% to 95% of their parents), in which 5 of them decreased significantly. For 3 transgenic lines of Xinong 889, their exogenous gene copy number was 2, 6 and 8, respectively, and their corresponding lipoxygenase activity was 32.76%, 26.39% and 21.05% of their parent, respectively. For 3 transgenic lines of Shanyou 225, their exogenous gene copy number was 1, 3 and 6, respectively, while their corresponding lipoxygenase activity was 95.47%, 51.06% and 37.73% of their parent, respectively. The copy number of exogenous Loxi in the transgenic wheat lines was negatively correlated with the lipoxygenase activity in its the seeds. The results showed that the activity of lipoxygenase in these transgenic wheat lines could be suppressed effectively by RNAi. In this study, the transgenic wheat lines with low lipoxygenase activity in their seeds were developed, and their exogenous gene copy number was analyzed. These transgenic wheat lines with very low lipoxygenase activity in their seeds can be used as parent marterials or new germplasm in wheat breeding program.
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Received: 23 January 2014
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SHEN Xue-Jing, ZHANG Er-Qin, XU Qian-Ru, LIU Lin-Ke, WAN Bo, LIU Yun-Chao, SUN Ya-Ning, ZHAO Dong, NIU Xiang-Xiang, DENG Rui-Guang, ZHANG Gai-Ping. Expression of Rabies virus G Protein in Rice (Oryza sativa) and Identification of Its Genetic Stability[J]. 农业生物技术学报, 2019, 27(2): 204-211. |
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